Structural insights into flexible pyruvate binding in an (S)-selective ω-transaminase.

Area of Science:

• Biocatalysis and enzyme engineering
• Structural biology and enzymology
• Organic chemistry and stereoselective synthesis

Background:

• (S)-selective ω-transaminases (S-ωTAs) are crucial biocatalysts for synthesizing chiral amines from ketones.
• The precise mechanisms of substrate recognition and active site dynamics in S-ωTAs remain incompletely understood.
• Understanding these mechanisms is key to optimizing enzyme performance for industrial applications.

Purpose of the Study:

• To elucidate the structural basis of keto-acceptor recognition in S-ωTAs.
• To investigate the active site flexibility and substrate binding pathway.
• To provide a structural framework for rational enzyme engineering of S-ωTAs.

Main Methods:

• X-ray crystallography of a marine S-ωTA (OM-S25) complexed with pyridoxal-5'-phosphate (PLP) and pyruvate (PYR) at 1.96 Å resolution.
• Molecular docking simulations to corroborate substrate binding poses.
• Enzyme activity assays with pyruvate derivatives and thermal shift assays.
• Site-directed mutagenesis of key active site residues.

Main Results:

• The crystal structure revealed significant conformational heterogeneity in PYR binding across enzyme protomers.
• Identified distinct PYR binding poses, including channel-proximal transitional states and deeply buried productive poses.
• Proposed a stepwise PYR entry pathway involving initial capture by Lys166, rotation, and stepwise relocation within the active site.
• A conserved arginine residue acts as an 'arginine switch' to stabilize PYR carboxylate groups.

Conclusions:

• The study provides unprecedented structural insights into acceptor recognition and binding dynamics in S-ωTAs.
• The proposed stepwise entry pathway offers a mechanistic model for the transamination second half-reaction.
• These findings are foundational for future mechanistic studies and the rational design of improved S-ωTAs for biocatalysis.